Treatment of cataracts is the single largest expense item in the U.S. Medicare budget, costing over $5 billion a year and affecting about 8 million Americans. There are over twenty different causes of cataracts and, although surgical treatment of the disorder is effective, there are no more conservative or less expensive therapeutic alternatives at this time. Additionally, many patients throughout the world do not have access to surgical treatments for this disorder.
A clear understanding of the pathogenesis of the disorder, which affects the same population of people as does Alzheimer's disease, is lacking. Cataracts and Alzheimer's disease may be linked by a rise in lenticular copper concentrations as a stochastic consequence of aging which is a common risk factor for both disorders. Several groups have observed that copper levels are elevated on the order of 50-fold in the cataract-affected eye and in the cataractous lens itself (Cekic, O., Br. J. Opthal. 82:186–188 (1998)).
A substantial body of evidence has accumulated suggesting that oxidative processes play a prominent role in the cascade of biochemical events leading to cataract formation (Spector, A., Ciba Foundation Symposium 106:48–62 (1984)), macular degeneration and retinitis pigmentosa. These oxidative processes are the end result, the “downstream” final common biochemical pathway, of cellular damage. The chemical reactions that involve redox-active metals (such as copper and iron) and oxygen, result in free radical species which are known to be toxic to most cells in living tissue, including the eye. The end-products of these chemical reactions are known as reactive oxygen species (ROS) and include hydrogen peroxide, superoxide anion, singlet oxygen, and the highly reactive and toxic hydroxyl radical. ROS are known to toxically interact with cellular proteins, nucleic acids, lipid membranes, and other essential cellular constituents, resulting in cross-linking and/or degradation and ultimately leading to cell damage and death. As a result of these processes, the functional integrity of the tissues so affected is compromised. Over the course of a lifetime of exposure to ROS, biological systems deteriorate, ultimately leading to degenerative or frank disease states.
In cataracts, the long-lived lenticular crystallin proteins accumulate post-translational chemical modifications (e.g., proteolytic fragmentation, glycation, amino acid racemization, disulfide and covalent cross-linking, carbonylation, and methionine oxidation, among others) and form high molecular weight protein cross-linked aggregates within the lens, specifically within the cytosol. Many of these changes are suspected to be the direct result of exposure to ROS and may lead to profound alterations in protein conformation. Thus, during cataractogenesis, α-crystallin undergoes a conformational transition from a soluble protein found in the transparent lens to a colored, insoluble, highly cross-linked aggregate (Chen, Y. C. et al., Exp. Eye Res. 65: 835–840 (1997); Harding, J. J., Biochem J. 129: 97–100 (1972); Harding, J. J., Curr. Opin. Ophthalmol. 9: 10–13 (1998); Dilley, K. J., and Pirie, A., Exp. Eye Res. 19: 59–72 (1974)).
As the crystallin proteins are not susceptible to protein clearance mechanisms in the fiber cells in the interior of the lens, the modified and aggregated crystallin protein masses accumulate (“condense”—Benedek, G. B., Invest. Ophthal. Vis. Sci. 38:1911–1921 (1997)) in an increasingly disordered fashion, leading one prominent researcher to place cataracts within the framework of conformational diseases (Carrell, R. W. and Lomas, D. A., Lancet 350:134–138 (1997)) such as Alzheimer's disease, sickle-cell anemia, and Creutzfeld-Jakob disease (Harding, J., J. Curr. Opin. Ophthalmol. 9:10–13 (1998)). This oxidatively engendered protein cross-linkage and aggregation results in progressive opacification of the lens (the sine qua non of cataracts) with decreased light transmission to the retina, and increased light scattering within the lens itself. The combination of these processes leads to blindness.
Evidence suggesting that oxidative processes are involved in cataractoge nesis is consistent with clinical evidence demonstrating increased hydrogen peroxide levels in the aqueous humor of cataractous eyes, increased lipid peroxidation markers such as malonidaldehyde in aged and cataractous lenses, and decreased antioxidant in cataractous lens (Bhuyan, K. C. et al., Life Sci. 38: 1463–1471 (1986); Micelli-Ferrari, T. et al., Br. J. Ophthalnol. 80: 840–843 (1996); Spector, A., Ciba Foundation Symposium 106:48–64 (1984); Ramachandran, S. et al., Exp. Eye Res. 53: 503–506 (1991)). As noted above, numerous studies have also demonstrated elevated levels of total copper in cataractous lenses (Cekic, O., Br. J. Ophthalmol. 82: 186–188 (1998); Balaji, M. et al., Br. J. Ophthalmol. 76: 668–669 (1992); Rasi, V. et al., Ann. Ophthalmol. 24: 459–464 (1992); Srivastava, V. K. et al., Acta Ophthalmol. (Copenh.) 70: 839–841 (1992); Racz, P., and Erdohelyi, A., Ophthalmic. Res. 20: 10–13 (1988); Cook, C. S., and McGahan, M. C., Curr. Eye Res. 5: 69–76 (1986); Nath, R. et al., Indian J. Exp. Biol. 7: 25–26 (1969); Srivastava, V. K. et al., Acta Ophthalmol., 70:839–841 (1992); Obara, Y., Nippon Ganka Gakkai Zasshi, 99:1303–1341 (1995)). This finding is important as Cu(II) is a co-factor in generating potentially damaging ROS, such as hydrogen peroxide and superoxide, which may foster protein aggregation as noted in other systems (e.g., the Alzheimer's disease Aβ1-42 protein) (Huang, X. et al., Biochem. 38: 7609–7616 (1999)). Further, decreases in the level of antioxidant defense enzymes such as glutathione reductase, glutathione peroxidase and superoxide dismutase, as well as decreases in total glutathione and corresponding increases in oxidized glutathione, have been observed (Rogers, K. M., and Augusteyn, R. C., Exp. Eye Res. 27: 719–721 (1978); Fecondo, J. V., and Augusteyn, R. C., Exp. Eye Res. 36: 15–23 (1983); Bhuyan, K. C. et al., Life Sci. 38:1463–1471 (1986)).
Clinical efficacy of antioxidants such as vitamins A, C and E in delaying cataract formation provide further suggestive evidence of oxidative mechanisms in this disorder (Brown, N. A. P. et al., Eye 12:127–133 (1998); Beebe, D. C., Invest. Ophthalmol. Vis. Sci., 39:1531–1534 (1998)). Additionally, antioxidants such as ascorbate, vitamin E and pyruvate have been shown to protect against cataract formation in mice (Shambhu, D. V., Am. J. Clin. Nutr. 53:335S-345S (1991)).
Redox-active transition metals are involved in harmful oxidative processes associated with a number of disorders such as Huntington's Disease (Reynolds, et al., Lancet 2:979–980 (1989); Pearson et al., Neurosci. Lett. 144:199–201 (1992)); Parkinson's disease (Ogawa et al., Neurology 42:1702–1706 (1992)); HIV encephalopathy (Sarder et al., J. Neurochem. 64:932–935 (1995)); cerebral malaria (Sanni et al., Am. J. Pathol. 152:611–619 (1998)); and fetomaternal tolerance (Sanni et al., Science 281:1191–1193 (1998)). The possible significance of redox-active metal in cataractogenesis is highlighted by the well-known clinical observation of rapid cataract formation following introduction of intraocular foreign bodies containing transitional metals such as copper or iron.
Thus, there is a need to find therapeutic agents that will inhibit or disrupt the various processes that are involved in cataract formation and development. Assay methods are needed that may be used to screen the many existing compounds, and compounds yet to be created, for their ability to disrupt the oxidation reactions and related cross-linking reactions that lead to the formation of cataracts, or to find molecules that retard or delay the progression of vision loss due to such cataract-causing reactions.